Opioid receptor delta as a global modulator of skin differentiation and barrier function repair.

OBJECTIVES: The aims of this study were to confirm the properties of selective agonist peptide (Rubixyl) contained in the spinach towards opioid receptor delta. In fact, agonist properties of both spinach peptides (Rubiscolin-5 and Rubixyl) towards opioid receptor delta were demonstrated by Zang et al., but their effects on the other opioid receptors were not studied [1]. We also studied the expression of opioid receptor delta in epidermis under normal and stress condition (inflammatory) and its role in epidermis homeostasis under stress condition in vitro and in vivo. METHODS: Agonist properties studies were performed using functional agonist cellular model containing human opioid receptors. Opioid receptor delta expression and epidermis homeostasis were studied on human reconstructed epidermis under normal and stress conditions (inflammatory stress) using gene expression (RT-qPCR) and protein expression analysis (immunohistological analysis). Skin repair properties of opioid receptor delta agonist were based on the following parameters TEWL (trans epidermal water loss, hydration and wrinkle depth at periocular and perilabial area) on human volunteers having either intrinsic ageing (more than 40 years old and non-smoker group) and both intrinsic ageing and extrinsic ageing (more than 40 years old and smoker group). RESULTS: We have demonstrated that the Rubixyl peptide is a specific agonist of opioid receptor delta. We have demonstrated that opioid receptor delta expression is modulated under inflammatory condition. The agonist Rubixyl was able to block the depletion of opioid receptor delta seen under inflammatory condition in reconstructed human epidermis. Inflammatory conditions lead to the unbalanced gene and protein expressions of markers involved in epidermis integrity and barrier function properties. The treatment of human reconstructed epidermis with the agonist Rubixyl leads to the normalization of unbalanced gene and protein expressions. In vivo study has confirmed the efficiency of the agonist Rubixyl to repair damaged skin by decreasing TEWL, increasing hydration and decreasing wrinkle depth at the periocular and perilabial area. CONCLUSION: In this research, we have demonstrated in vitro (on inflamed reconstructed human epidermis, RHE) and in vivo (on human aged volunteers) that activation by natural agonist peptide of opioid receptor delta reduces the skin inflammation thus leading to improvement in epidermis differentiation and skin barrier properties.

Improvement of polyphenol properties upon glucosylation in a UV-induced skin cell ageing model.

OBJECTIVE: Polyphenols are strong antioxidant molecules allowing prevention of skin photo-ageing damages, but their use is limited due to low solubility and toxicity towards skin cells. We postulated that enzymatic glucosylation could improve their solubility, stability and, consequently, their efficacy. The aim of this work was to study changes induced by addition of a glucose moiety on two polyphenols displaying very different chemical structures [caffeic acid (CA), epigallocatechin-3-gallate (EGCG) and there glucosylated form, Glc-CA and Glc-EGCG] by assessing their cytotoxic properties and their antioxidant and anti-inflammatory activities. METHODS: Their antioxidant effect was assessed first by the classical DPPH radical-scavenging method. Then, a panel of human skin cells (keratinocytes, melanocytes, fibroblasts and endothelial cells) was used to evaluate their effect on cell toxicity and their antioxidant activities. With this aim, a photo-ageing model based on UV irradiation of skin cells was established. Molecule activity was assessed on reactive oxygen species (ROS) production, on superoxide dismutase (SOD) and catalase activities and, finally, on inflammatory factor production IL-6, IL-8 and IL-1ß. RESULTS: In an acellular model, antioxidant activity assessed by DPPH method was strongly reduced for Glc-CA compared to CA, whereas it remained the same for Glc-EGCG compared to EGCG. Glucosylated derivatives did not display more toxic effect on various skin cells. Moreover, toxicity was even strongly reduced for caffeic acid upon glucosylation. The efficacy of glucosyl-compounds against UV-induced ROS production was preserved, both with pre- and post-UV treatments. Particularly, a better antioxidant efficacy was shown by Glc-EGCG, vs. EGCG, on keratinocytes. In addition, an induction of SOD and catalase activity was clearly observed for Glc-CA. Both glucosyl-polyphenols display the same activity as their parent molecule in decreasing inflammatory factor production. CONCLUSION: Our results demonstrated that enzymatic glucosylation of CA and EGCG led to an improved or preserved antioxidant activity in a cellular model of UV-induced skin ageing, despite the decrease in instantaneous antioxidant properties observed for Glc-CA. Glc-EGCG is specifically more active on keratinocytes, suggesting a specific targeting. Such glucosylated polyphenols displaying improved physicochemical and biological properties should be better candidates than natural ones for use in food additives and cosmetics.

[Liposomes and skin: past, present, future]. / Liposomes et peau: passé, présent, futur.

tAmong various approaches to intra- and percutaneous administration of drugs, e.g. application of patches, ointments, iontophoresis, electroporation, the use of lipid vesicles like liposomes and niosomes presents numerous advantages. They are not toxic or invasive, may deliver hydrophobic and/or hydrophilic molecules, and the size of the transported molecule is not a limiting factor. Liposomes are obtained with natural amphiphilic lipids whereas niosomes are composed of synthetic amphiphilic molecules. These microscopic vesicles contain from one to several concentric lipid bi-layers with intercalated aqueous compartments. Trans-epidermal penetration of the vesicles is proportional to the "fluidity" of their lipids and their negative charge. Several drugs and cosmetics in this gallenic form are already commercially available and successfully used, presenting a better dose/effect ratio and provoking less side-effects.

In vitro evaluation of the sensitization potential of weak contact allergens using langerhans-like dendritic cells and autologous T cells.

Contact hypersensitivity is a major public health concern in most industrial countries, which is why predictive tests which could identify potential allergens are needed. We have established an in vitro approach for the detection of primary immune response. This model uses Langerhans-like dendritic cells (LLDC) derived from cord blood progenitors and autologous T lymphocytes, isolated from the same blood sample. Treatment of day 12-14 LLDC, with strong haptens trinitrobenzene sulfonic acid (TNP), fluorescein isothiocyanate (FITC) or Bandrowski's base (BB), results in the proliferation of T lymphocytes, whereas weak allergens and irritants, such as sodium dodecyl sulfate (SDS) are ineffective. The use of immature (day 8) LLDC and the addition of a 48 h stage of incubation after hapten contact, result in phenotypic maturation of LLDC in addition to lymphocyte activation in all the cultures with strong haptens. The 48 h stage of incubation, results in sensitization and in some cases the induction of T cell proliferation to citronellal (1/8), coumarine (1/8) and to a prohapten p-phenylenediamine (pPDA; 2/8). The phenotype of DC after 48 h of contact with a strong hapten, becomes that of mature DC (CD83(+), CD86(+) and HLA-DR(++)). With fragrance molecules, weak haptens and prohaptens, a comparable phenotype is observed only when T lymphocytes are activated. These data suggest that the unresponsiveness observed with weak haptens, may be the consequence on an incomplete maturation of LLDC.

Evaluation of the capacity of dendritic cells derived from cord blood CD34+ precursors to present haptens to unsensitized autologous T cells in vitro.

Langerhans cells play a key role in contact hypersensitivity reactions. The application of haptens on the skin leads to many modifications of these cells, including the increase of major histocompatibility complex II expression, allogeneic stimulation potency, and migration towards lymph nodes to activate T cells. Moreover, it has been shown that Langerhans cells cultured in vitro are able to prime naive T cells in response to hapten contact. From CD34+ progenitors present in cord blood, we generated dendritic cells of which some presented the phenotypic markers of Langerhans cells. We show that these cells are able to sensitize syngeneic naive (CD45RA+) T cells to haptens such as trinitrophenyl conjugate of trinitrobenzene sulfonic acid (TNP) and fluoroscein isothiocyanate. The response to TNP is higher than to fluoroscein isothiocyanate, whereas sodium dodecyl sulfate, an irritant molecule used as a control, never caused this effect. Phenotypic analysis of cellular suspensions and experiments of cell sorting lead to the conclusion that only CD1a+ cells are able to induce a primary response of syngeneic T cells to TNP or fluoroscein isothiocyanate. Furthermore, we have shown a close relationship between the differentiation state of dendritic cells and their ability to prime T lymphocytes. Dendritic cells are able to present haptens in an efficient manner between day 10 and 14 of culturing CD34+ progenitors, whereas they were efficient in presenting alloantigens from day 6 until after day 20. This dissociation suggests the need of an active metabolic process for hapten presentation in the direct treatment of dendritic cells with haptens. This model of hapten presentation was used for a panel of fragrance molecules and other molecules considered as weaker haptens than TNP and fluoroscein isothiocyanate.

Ultraviolet rays induced expression of lectins on the surface of a squamous carcinoma keratinocyte cell line.

Human keratinocytic cells from squamous carcinoma (SCL-1) present, under resting conditions, relatively low amounts of endogenous lectins (sugar-binding proteins). Upon uv irradiation, they express on their cell surface large amounts of endogenous lectin molecules able to bind neoglycoproteins bearing either alpha-L-rhamnosyl or alpha-D-glucosyl residues. A similar binding specificity was found with normal human keratinocytes under the same culture conditions. At sunlike doses, uv.A (365 nm) was more efficient than uv.B (312 nm) in the expression of such receptors on the surface of SCL-1 cells. The increased presentation of lectins by SCL-1 cells was transient and reached a maximum 4 h after irradiation. Such a specific modulation of receptor expression upon uv irradiation might be biologically significant, considering the numerous intercellular recognition phenomena in skin biology. alpha-L-Rhamnose-specific receptor on SCL-1 could not be distinguished from alpha-D-glucose-specific receptor on the basis of neoglycoproteins binding, uptake, and related inhibitions. Lectin expression was mainly detected on the cell surface, and its overexpression due to uv rays required a de novo protein synthesis process.

In vitro primary sensitization of hapten-specific T cells by cultured human epidermal Langerhans cells--a screening predictive assay for contact sensitizers.

BACKGROUND: The need to develop predictive tests which could identify potential allergens has been recognized for many years. There is as yet no accepted in vitro method for the assessment of contact sensitizers. OBJECTIVE: We have tested the ability of a range of contact allergens to induce in vitro primary sensitization of autologous T cells. METHOD: T-cell proliferation induced by haptens using 2-day cultured human Langerhans cells as antigen-presenting cell was assessed by 3H thymidine incorporation. Antigen specific stimulation was calculated as stimulation indexes. RESULTS: Strong allergens induced in vitro a primary T-cell response in all (trinitrophenyl, TNP: 13/13) or in the majority (fluorescein isothiocyanate, FITC: 7/10) of experiments. An irritant, sodium dodecyl sulfate (SDS), failed to generate a significant T-cell proliferation in any of the experiments (0/10). We obtained a significant lymphoproliferative response to weak sensitizers only in a limited number of experiments: (coumarin: 1/12, citronellal: 0/10, hydroxycitronellal: 2/8). p-Phenylenediamine (PPDA), a prohapten and highly sensitizing chemical in vivo, generated primary sensitization in vitro in only one of six experiments, while Bandrowski's base (BB), a metabolization product of PPDA induced a significant T-cell response in all six experiments. CONCLUSION: The present in vitro model allows discrimination between two groups of substances: strong contact sensitizers (TNP, FITC, BB) on the one hand and weak sensitizers (coumarin, citronellal and hydroxycitronellal) and irritants (SDS) on the other hand. It could be used as a screening in vitro assay to eliminate strong contact allergens before further predictive animal tests have to be performed.

Expression and function of B7-1 (CD80) and B7-2 (CD86) on human epidermal Langerhans cells.

In addition to T cell receptor triggering, activation of T cells requires costimulatory signals that have been shown to be mainly initiated through CD28. We analyzed the expression and function of the two ligands for CD28, B7-1 (CD80) and B7-2 (CD86), on human Langerhans cells (LC), the antigen-presenting cells from epidermis. Human LC freshly isolated from epidermis (fLC) expressed significant level of B7-2, which was increased upon a short culture in vitro. In contrast, B7-1 was undetectable on fLC but appeared at the cell surface after a 3-day culture in vitro. Pre-incubation of 18-h cultured LC with anti-B7-2 monoclonal antibodies (mAB) was sufficient to abrogate the binding of CTLA4-Ig fusion protein, while a combination of both mAB against B7-1 and B7-2 was necessary to obtain a complete inhibition of CTLA4-Ig binding on 3-day cultured LC, showing the absence of a third CTLA4 ligand. The function of B7-1 and B7-2 on human LC has been analyzed by adding mAb at the beginning of mixed epidermal cell lymphocyte reactions. Anti-B7-2 mAb and CTLA4-Ig, but not anti-B7-1 mAb, strongly inhibited allogenic. as well as recall antigen-induced T cell proliferation supported by fLC or 3-day cultured LC. Collectively, these results demonstrate that B7-2 is the major ligand for CD28/CTLA4 at the LC surface and that it plays a crucial role in human LC co-stimulatory function with little, if any, dependence of B7-1 expression.

cis-urocanic acid failed to affect in vitro human Langerhans cell allostimulatory function.

Urocanic acid (UCA) represents the major ultraviolet B (UVB, 290-320 nm)-absorbing component of the skin. Trans-UCA is naturally produced in the stratum corneum and converts to the cis isomer upon UVB irradiation. In this study, we examined the effect of purified cis-UCA (about 99% of cis isomer) on the human Langerhans cell (LC) allostimulatory function by using the mixed epidermal cell-lymphocyte reaction (MELR). We found that addition of increasing amounts (6.5-400 micrograms/mL) of purified cis-UCA or trans-UCA did not modify the T-cell response supported by enriched LC (eLC: 8-25% LC) as well as purified LC (pLC: 70-90% LC) suspensions. Because cis-UCA had no effect on the allostimulatory function of untreated LC, we investigated whether this compound could modify T-cell proliferation induced by UVB-irradiated LC. The UVB exposure of eLC or pLC to 100 J/m2 significantly inhibited the capacity of both suspensions to mount a T-cell response. However, addition of cis-UCA did not potentiate this UVB-induced immunosuppression. The eLC or pLC were then incubated with cis-UCA for 18 h at 37 degrees C and washed before adding to allogeneic T cells. The obtained proliferative response was similar to that induced by control LC incubated in medium alone, demonstrating that pretreatment with cis-UCA did not alter human LC function. In conclusion, these results strongly suggest that cis-UCA has no direct effect on human LC antigen-presenting function.

In vitro effects of ultraviolet B radiation on human Langerhans cell antigen-presenting function.

The effects of ultraviolet B radiation (UBV) on the immune function of human epidermal Langerhans cells (LC) were studied by using the mixed epidermal cell-lymphocyte reaction (MELR). Exposure of both enriched LC suspensions (eLC, 8-20% LC) and purified LC suspensions (pLC, 70-90% LC) to increasing doses of UVB radiation (25 to 200 J/m2) decreased the proliferative T cell response in a very similar dose-dependent way, suggesting that keratinocytes did not play a major role in the UVB-induced inhibition of MELR. Supernatants from irradiated cultured eLC or pLC failed to inhibit T cell proliferation induced by untreated pLC. Furthermore, addition of irradiated eLC to untreated pLC did not affec the allogeneic T cell response. Taken together, these results provide evidence that in vitro UVB-induced immunosuppression was not mediated by inhibitory soluble factors that could affect either LC allostimulatory property or T cell proliferative response. UVB irradiation of human LC inhibited the capacity of these cells to induce CD4+ as well as CD8+ T cell proliferation. UVB-irradiated LC also induced a decreased T cell response to recall antigen or mitogen. Moreover, addition of exogeneous cytokines such as IL-1 beta, IL-1 alpha, or IL-2 did not reverse the defective function of UVB-irradiated LC in MELR. The inhibitory effect of UVB radiation on human LC was not related to a decreased HLA-DR expression. Because cultured LC appeared to be less sensitive than freshly isolated LC to UVB-induced suppressive effects, the deleterious effects of UVB radiation on human LC allostimulatory properties may be associated with an impaired development of LC accessory function.

Lovastatin enhances the photocytotoxicity of UVA radiation towards cultured N.C.T.C. 2544 human keratinocytes: prevention by cholesterol supplementation and by a cathepsin inhibitor.

The effect of the hydroxymethylglutaryl-CoA (HMG-CoA) inhibitor lovastatin on the UVA-induced photocytotoxicity has been investigated in cultured human N.C.T.C. 2544 keratinocytes. In the absence of irradiation, 5 x 10(-7) M lovastatin did not exhibit any significant cytotoxic effect towards this cell line. Although the drug cannot act as a photosensitizer, because it does not absorb in the UVA range, it markedly increased the UVA-induced cellular damage (about 70% reduction in cell viability at 5 x 10(-7) M). This effect was not accompanied by an increase in the lipid peroxidation product content of cells as compared with treatment with UVA alone. Medium supplementation with 0.01 mg/ml free cholesterol totally prevented the enhancement of UVA photocytotoxicity induced by lovastatin. A protective effect was also observed when cells were supplemented with an amount of low-density lipoprotein giving the same cholesterol concentration in the culture medium. Finally, E64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane], a lysosomal cathepsin inhibitor, also prevents the cell death induced by UVA in cells treated with lovastatin. These results suggest that HMG-CoA reductase inhibitors could increase the sensitivity of skin cells to UVA radiation, and that this phenomenon is related to lysosomal enzyme release.

Polyunsaturated fatty acid enrichment increases ultraviolet A-induced lipid peroxidation in NCTC 2544 human keratinocytes.

The influence of cell enrichment with fatty acids with increasing degree of unsaturation on the ultraviolet A-induced formation of lipid-peroxidation products (thiobarbituric acid reactive substances [TBARS]) has been investigated in NCTC 2544 human keratinocytes. A 48-h preculture of cells in controlled medium supplemented with unsaturated fatty acids resulted in a marked increase in TBARS appearance under ultraviolet A exposure. This effect was dependent upon the degree of unsaturation of the fatty acids, with the following order of efficiency: arachidonic > linolenic > linoleic > oleic acid. For arachidonic acid (AA), the potentiating effect on ultraviolet A-induced lipid peroxidation was dependent upon the fatty acid concentration, with about a 2.5-fold increase in TBARS formation in cells pre-cultured with 5 x 10(-5) M AA, then exposed to a UVA dose of 13 J/cm2. The increase in TBARS formation by AA was almost totally prevented by supplementation of cells with 5 x 10(-5) M vitamin E, whereas buthionine sulfoximine, a chemical which depletes cell glutathione, potentiated lipid peroxidation. These results suggest that the nature of the fatty acids of cellular lipids could influence the response of keratinocytes to ultraviolet A, and especially the ultraviolet A-induced lipid peroxidation.

In vitro primary sensitization and restimulation of hapten-specific T cells by fresh and cultured human epidermal Langerhans' cells.

We examined the capacity of human Langerhans's cells (LC) to sensitize autologous T cells to the trinitrophenyl hapten (TNP) in vitro. Two-day cultured Langerhans' cells, but not freshly prepared Langerhans' cells, can induce in vitro primary proliferative reactions to the TNP hapten. Using a CD45RA+ naive T-cell subset, similar results were found, therefore making the possibility of a previous in vivo T-cell contact with the hapten unlikely. The primary in vitro response was strongly inhibited by monoclonal antibodies to major histocompatibility complex (MHC) class I and II, CD4 antigens and ICAM-1 and LFA-3 adhesion molecules. Furthermore, we found that fresh LC can prime T cells to TNP, as revealed by a significant secondary T-cell proliferation after restimulation of the recovered T lymphocytes by fresh hapten-modified autologous LC. Nevertheless, the ability of these fresh LC to stimulate in vitro secondary hapten-specific T-cell proliferation was very limited in comparison with that of 2-day incubated Langerhans' cells. After secondary stimulation with TNP-cultured LC, sensitized T cells could be non-specifically expanded without losing hapten specificity. The TNP-specific T-cell lines were mostly of the CD4+ phenotype. The present findings extend previous studies in the mouse, showing that culture LC are potent antigen-presenting cells (APC) in primary hapten-dependent proliferation assays. Furthermore, this in vitro priming assay, using cultured human Langerhans' cells as APC, might be useful to analyse the early steps of T-cell sensitization and subsequently to develop in vitro predictive tests allowing detection of sensitizing compounds.

C32 human melanoma cell endogenous lectins: characterization and implication in vesicle-mediated melanin transfer to keratinocytes.

To optimize skin pigmentation in order to help body prevention against UV radiation, the mechanism of melanin pigment transfer from melanocytes to keratinocytes must be elucidated. Melanin transfer to keratinocytes requires specific recognition between keratinocytes and melanocytes or melanosomes. Cell surface sugar-specific receptor (membrane lectin) expression was studied in human C32 melanoma cells, an amelanotic melanoma, by flow cytometry analysis of neoglycoprotein binding as an approach to the molecular specificity. Sugar receptors on melanocytes are mainly specific for alpha-L-fucose. Their expression is enhanced upon treatment by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which can induce melanin synthesis in amelanotic human melanoma cells in a dose-dependent manner. Flow cytometry analyses showed a small-sized population of vesicles distinguishable from large cells by their fluorescence properties upon neoglycoprotein binding. Sorting indicated that the small-sized subpopulation is composed of vesicles produced by melanocytic cells. Upon vesicle formation, a selective concentration of sugar receptors specific for 6-phospho-beta-D-galactosides appears in the resulting melanocytic vesicles. Vesicles are recognized and taken up by cultured keratinocytes and a partial inhibitory effect was obtained upon cell incubation in the presence of neoglycoproteins, indicating a possible participation of sugar receptors in this recognition. The validity for such a model to help in understanding the natural melanin transfer by melanosomes is confirmed by electron microscopy, which demonstrates the presence of melanin inside keratinocytic cells upon incubation with melanocytic vesicles.

Human keratinocyte membrane lectins: characterization and modulation of their expression by cytokines.

In an attempt to identify cell surface molecules involved in recognition phenomena between cells such as keratinocytes and melanocytes and putatively target biological responses modifiers to keratinocytes, we undertook the detection of cell surface sugar specific receptors: membrane lectins. Keratinocyte membrane lectins were found to bind synthetic glycoproteins (neoglycoproteins) carrying either alpha-L-fucosyl or alpha-L-rhamnosyl residues. Fluorescence microscopy observations indicate that cultured keratinocytes are able to bind these two neoglycoproteins while frozen sections of human skin labelled with neoglycoprotein-coated covaspheres show that the selectivity of the binding to keratinocytes is restricted to alpha-L-rhamnosyl-BSA. Keratinocytes were adapted to grow on collagen; harvesting conditions allowing the analysis of keratinocytes by flow cytometry are described. This technique allows the quantification of the binding at 4 degrees C, and the estimation of the endocytosis of F-, neoglycoproteins: F-, alpha-L-Rha-BSA and F-, alpha-L-Fuc-BSA were efficiently internalized. Thereafter, alpha-L-rhamnose-substituted liposomes containing 5-(6)carboxyfluorescein were prepared in order to follow the delivery of the fluorescent dye into cells. This was measured both by flow cytometry and by spectrofluorimetry. The expression of surface lectins was checked upon action of cytokines (IL1 alpha, IL1 beta, IL2 and TNF) which are known as biological response modifiers of keratinocytes.

Modulation of the binding and endocytosis of concanavalin A by guinea pig keratinocytes: reversible antagonistic effects of cholesterol and phospholipid-liposomes.

Alteration of guinea pig keratinocyte membrane microviscosities (eta) by liposomes of varying composition was determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Measurements performed either with whole cell suspensions or Percoll-separated cell subpopulations, indicate a similar membrane microviscosity (eta = 3.37 poise +/- 10%) compared to those microviscosities reported for other cell types. Our findings show that treatment of guinea pig keratinocytes with liposomes composed of phospholipids results in a decreased membrane microviscosity (1.95 poise), whereas treatment of the cells with an emulsion of cholesterol hemisuccinate, or liposomes composed of cerebrosides, causes an increase in membrane microviscosity (3.85 poise and 5.55 poise +/- 10%, respectively). Changes in membrane fluidity had no significant effect on cell viability. A reduced membrane microviscosity resulted in a decrease in the binding of Concanavalin A to keratinocytes, whereas an increased microviscosity resulted in an increased binding of Concanavalin A. Furthermore, endocytosis of Concanavalin A bound to keratinocytes plasma membranes was not significantly affected by a reduced membrane microviscosity, whereas an increased membrane microviscosity completely blocked the endocytosis of Concanavalin A. Another novel observation was that membranes "fluidified" by phospholipid liposomes could be "rigidified" by treatment with cholesterol hemisuccinate and vice versa. Moreover, these alternate changes in membrane microviscosity resulted in simultaneous alternate changes in the binding of Concanavalin A to the keratinocyte surface.

Decreased biosynthesis of actin and cellular fibronectin during adipose conversion of 3T3-F442A cells. Reorganization of the cytoarchitecture and extracellular matrix fibronectin.

Differentiation of 3T3-F442A cells was accompanied by changes in cell morphology, decreased synthesis and assembly of actin and fibronectin. The network of microfilament stress fibers detected with NBD-phallacidin was altered during adipose conversion of 3T3-F442A cells. Parallel to this, the disappearance of fibrillar bundles of extracellular matrix fibronectin was observed by immunofluorescence staining. The pericellular fibronectin content, detected by immunoblotting, strongly diminished during the differentiation process. An altered rate of biosynthesis of both proteins was also measured by [35S]-methionine pulse-labeling and immunoprecipitation. A 4-5-fold decrease in cellular fibronectin synthesis was observed in adipocytes compared to control preadipocytes. Conversely, non-differentiating 3T3-C2 control cells did not reorganize either the cytoskeletal architecture or the extracellular matrix fibronectin in the resting state. These results suggest that the decreased rate of biosynthesis of cell-associated fibronectin is correlated with that of actin. Moreover, both events can essentially be ascribed to differentiation.

Growth and differentiation of 3T3-F442A preadipocytes in three-dimensional gels of native collagen.

Three-dimensional gels of native type I collagen have been used as a substrate for growth and differentiation in 3T3 adipocyte precursors. Such hydrated lattices can support a sustained cell growth leading to several 10-fold increases in cell number within 2 weeks. During this period, the cells condense the hydrated collagen lattice to a tissue-like structure one-fourth of the area of the initial gel. From Days 10 to 12, the cells progressively exhibit morphological characteristics of adipocytes and accumulate lipid droplets as evidenced by Oil Red O staining. Lipoprotein lipase activity appears very early; between Days 8 and 22 it sharply increases 15-fold and then remains stable at a very high level (about 30 nmol/min/10(6) cells). The emergence of glycerophosphate dehydrogenase activity is delayed; it becomes detectable at Day 15 and progressively increases up to 700 nmol/min/10(6) cells at Days 35-40. Thus, this adipose tissue equivalent appears to be a potential model for studying adipocyte function.